Electrophoresis is widely used for fractionation of a variety of biomolecules, including DNA species, proteins, peptides, and derivatized amino acids. One electrophoretic technique which allows rapid, high-resolution separation is capillary electrophoresis (CE) (Cohen, 1987, 1988, Compton, Kaspar, Lauer). Typically, CE employs fused silica capillary tubes whose inner diameters are between about 50-200 microns, and which can range in length between about 10-100 cm or more.
In the usual electrophoresis procedure, an electrophoresis tube, such as a capillary tube, is filled with a fluid electrophoresis medium, and the fluid medium is crosslinked or temperature-solidified within the tube to form a non-flowable, stabilized separation medium. A sample volume is drawn into or added to one end of the tube, and an electric field is applied across the tube to draw the sample through the medium. Electrophoretic separation within the matrix may be based on molecular size, in the case of nucleic acid species (which have roughly the same charge density), or on a combination of size and charge, in the case of peptides and proteins.
The polymer concentration and/or degree of cross-linking of the separation medium may be varied to provide separation of species over a wide range of molecular weights and charges. For separating nucleic acid fragments greater than about 1,000 bases, for example, one preferred temperature-solidified material is agarose, where the concentration of the agarose may vary from about 0.3%, for separating fragments in the 5-60 kilobase size range, up to about 2%, for separating fragments in the 100-3,000 basepair range (Maniatis). Smaller size fragments, typically less than about 1,000 basepairs, are usually separated in cross-linked polyacrylamide. The concentration of acrylamide polymer can range from about 3.5%, for separating fragments in the 100-1,000 basepair range, up to about 20%, for achieving separation in the size range 10-100 basepairs. For separating proteins, crosslinked polyacrylamide at concentrations between about 3-20 percent are generally suitable. In general, the smaller the molecular species to be fractionated, the higher the concentration of crosslinked polymer.
The resolution obtainable in solidified electrophoresis media of the type described above has been limited, in the case of small molecular weight species, by difficulties in forming a homogeneous, uniform polymer matrix at high polymer concentration within an electrophoresis tube, and especially within a capillary tube. In the usual method for forming a high-concentration solidified matrix in a capillary tube, the gel is produced inside the capillary by mixing the gel precursors (typically including reactive monomers or prepolymers, one or more crosslinking agents, polymerization catalyst, polymerization initiator and other additives that may be useful during the separation process, such as surfactants and denaturizers), filling the capillary with this mixture and allowing the gel to cure within the capillary. Unfortunately, in addition to the gel, this process leaves gel residues that can interfere with the chromatographic separation of sample components and lead to premature breakdown of the gel. Because of the extreme length-to-diameter ratio of capillary 15, these residues are not easily removed from the gel by flow of eluent through the capillary. Indeed, in electrophoresis, there is almost no flow of eluent through the gel. The only species that exhibit significant mobility are the ionic species.
Another problem is that the gel generally shrinks by a few percent volume when it cures, so that the gel tends to pull away from the walls of the capillary. As a result, when the electric field is turned on to push sample ions through the capillary, the gel tends to be pushed along and out of the capillary due to ionic groups associated with the gel. To prevent this, it is common to treat the inside surface of the capillary wall and/or to add to the gel precursor a coupling agent, such as silane, to bond the gel to the capillary wall.
An additional problem is that voids sometimes occur in the gel. Such voids are more readily produced in gels that are bonded to the capillary or column wall because they are prevented from pulling away from the wall as they shrink during curing. These voids present obstacles to the ionic flow and can introduce inhomogeneities in the process that degrade resolution. If such a void extends entirely across the internal diameter of the capillary, there will be a complete break in the current path and electrophoresis will be stopped.
To overcome the above problems, in one gel formation process, a capillary is first filled with the gel precursor. Preferable, the gel precursor is at a reduced temperature that inhibits the chemical reaction that results in the formation of the gel. The capillary is then either heated or exposed to radiation in a narrow zone to cure the gel precursor within that zone. This zone is then moved along the capillary to cur the gel along the entire length of the capillary. By use of this moving zone of curing, the still-mobile gel precursor can flow toward the cured zone to compensate for the shrinkage that occurs during curing. Unfortunately, this moving zone process is a slow process that is difficult to control and that significantly increases the time required to produce a capillary gel.
As another approach, it has been proposed to reduce void formation and gel shrinkage by carrying out the gel crosslinking reaction at high pressure. This approach has been only partially successful in reducing inhomogeneities in the gel, and adds substantially to the cost and complexity of the electrophoresis procedure.
Alteratively, in the case of temperature-solidifying polymers, the polymer is introduced into an electrophoresis tube in a fluid form, then allowed to gel to a solid form by cooling within the gel. This approach, however, has limited application, since polymers, such as agar and agarose, which are known to have the necessary temperature-solidifying setting properties, are not effective for fractionating low molecular weight species, even at high polymer concentrations.
A second limitation associated with crosslinked or temperature solidified matrices is the difficulty in recovering fractionated molecular species within the matrix, after electrophoretic separation. In the case of a preparative-scale electrophoresis tube, the solidified matrix must be carefully separated from the walls of the tube before the matrix can be removed, a procedure which is virtually impossible with capillary tubes. Even after the matrix is removed, and the region of the matrix containing the desired molecular species is identified, the species of interest can be recovered from the matrix region only by a lengthy elution procedure, or by electrophoretic elution.